EP0238645A1 - Nouveaux systemes d'administration de medicaments a base de lipoproteines - Google Patents

Nouveaux systemes d'administration de medicaments a base de lipoproteines

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Publication number
EP0238645A1
EP0238645A1 EP86906200A EP86906200A EP0238645A1 EP 0238645 A1 EP0238645 A1 EP 0238645A1 EP 86906200 A EP86906200 A EP 86906200A EP 86906200 A EP86906200 A EP 86906200A EP 0238645 A1 EP0238645 A1 EP 0238645A1
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EP
European Patent Office
Prior art keywords
sequence
dna
ldl
protein
active ingredient
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EP86906200A
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German (de)
English (en)
Inventor
Andrew A. Protter
John P. Kane
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Biotechnology Research Partners Ltd
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Biotechnology Research Partners Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1275—Lipoproteins; Chylomicrons; Artificial HDL, LDL, VLDL, protein-free species thereof; Precursors thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the invention relates to preparations for effective, targeted drug delivery.
  • it relates to using the receptor-binding activity of certain apolipoproteins, the phospholipid-binding ability of amphipathic helices contained in apolipoproteins, or both, for target-specific delivery of active substances to vertebrates.
  • Efficient drug delivery demands that the active ingredient of the pharmaceutical or other composition be carried from its point of administration to its site of activity or processing without undue dilution in the digestive or circulatory system to undermine its effect. It is only common sense that unwanted side effects of a particular drug will be minimized if it is immediately directed to its site of desired activity rather than allowed to make its own way through the subject's corpus and exert effects in undesired locations. This is particularly important, or example, when toxic substances are used to destroy particular unwanted cells such as tumor cells. Most such agents are equally, or at least similarly, capable of destroying healthy tissue; to the extent they are allowed to roam undirected they may result in considerable damage.
  • liver many materials are processed or modified by the liver in order to place them in an effective role, and as a general proposition, the desired delivery to a particular target is mediated by preliminary delivery to the liver.
  • specific targeting agents may direct the active ingredient either to the ultimate site, or to the liver for intermediate handling.
  • CM chylomicrons
  • VLDL very low density lipoproteins
  • LDL low density lipoproteins
  • HDL high density lipoproteins
  • LDL fraction contains more than 80% apolipoprotein B (apoB) and some apolipoprotein E (apoE) . Both of these proteins appear to be involved in the binding of the LDL to specific receptors in the liver and in the binding of the LDL to receptors on various cultured cell lines.
  • apoB apolipoprotein B
  • apoE apolipoprotein E
  • the lipoproteins in general contain a lipid center surrounded by a monolayer containing phospholipids and the apolipoproteins. Regardless of the nature of the specific binding of the surface apolipoproteins to receptors, attempts have been made to utilize the lipoproteins as delivery vehicles for drugs by sequestering an active substance in the lipid fraction. It was demonstrated by Rudman, D. , et al. Pharmacol Exp Ther (1972) 180: 797, that various materials of pharmaceutical interest, including estradiol, testosterone, and pentobarbital, can be sequestered in the lipoprotein fraction. Reconstituted LDL has also been shown to be an effective vehicle for the delivery of fluorescent probes to cells for the purpose of labeling (Krieger, M. , et al, J Supramol Struct (1979) 10.: 467-478), and reconstituted LDL containing cytotoxic agents has been used to deliver these agents specifically to tumor cells in vitro
  • ApoE is a 299 residue protein of known amino acid sequence that is a component of several of the lipoproteins.
  • the binding domain of apoE has been studied in relation to the receptor sites (Innerarity, T.L., et al. J Biol Chem (1983) 258: 12341-12347; Weisgraber. K.H. , et al. ibid. 12348-12354) .
  • a number of isofor s of this protein have been found.
  • Apolipoprotein B (apoB) is much larger. When deprived of its lipid content, the protein appears to have a molecular weight of about 380,000. corresponding to almost 4000 amino acid residues. This protein appears to be present in two major forms, the larger peptide designated B-100, which is associated with hepatic lipoproteins. and a somewhat smaller form designated B-48, which is associated with intestinal lipoproteins and the chylomicrons. The hepatic form, B-100, appears to be processed by proteolytic enzymes in the plasma into two fragments designated B-74 and B-26.
  • the receptor is an 839 amino acid protein having five identified domains, with the presumed ligand binding site consisting of a 322 amino acid cysteine-rich region containing eight negatively charged side chains.
  • apolipoproteins in general appear to contain specialized, but generic, structures to enable them effectively to bind phospholipids. These structures are considered to be amphipathic alpha helices which contain oppositely facing hydrophobic and hydrophilic regions (Sparrow, J.T., et al.
  • compositions and methods of the present invention provide new approaches to targeted drug delivery using the mediation of lipid transport.
  • the invention provides drug delivery systems which are both specifically targeted and lipid compatible.
  • the approach is two-fold,
  • the receptor recognition domains of apolipoproteins in particular of apolipoprotein B or E, are used to serve as a binding moiety, and are covalently linked to additional substances which are either carriers or themselves active ingredients.
  • ⁇ helical amphipathic regions such as those of apolipoproteins are used to bind directly to hydrophobic active ingredients or to bind lipid compatible carriers containing active ingredients. These amphipathic regions are in turn bound to specific targeting substances including the receptor binding regions of apoB or apoE as well as to completely different binding moieties such as antibodies.
  • a particularly preferred approach combines the receptor binding regions of the apolipoproteins with their amphipathic helices in a single protein which then serves as a directed carrier of a lipid soluble active ingredient.
  • the invention relates to a composition for delivery of an active substance which comprises an LDL receptor binding region such as that of apoB or apoE covalently bound to a substance which is either itself an active ingredient or a carrier for an active ingredient.
  • the invention is directed to a composition for delivery of an active substance which comprises a lipophilic peptide containing at least one amphipathic ⁇ helical domain conjugated covalently to a binding moiety, i.e., a compound that has specific binding regions, such as an im unoglobulin or an LDL r ceptor binding region.
  • the compositions and methods of the invention are suitable for use in any vertebrate subject including, and preferably, mammalian and human subjects.
  • compositions of the invention include methods of administering active ingredients employing the compositions of the invention and specific novel components of these compositions.
  • novel components include the LDL receptor binding region of the apoB protein and the amphipathic regions thereof, and fusion proteins comprised of LDL receptor binding region and amphipathic regions derived from alternate apolipoproteins. of amphipathic regions and specific binding moieties, such as antibodies, or of LDL receptor binding region and active substance.
  • the invention is directed to materials and methods useful in constructing the drug delivery compositions of the invention. including the relevant DNA sequences, vectors, expression systems, and transformed hosts.
  • Figure 1 shows the amino acid sequence of the N-terminal portion of apoB-26 with putative LDL-receptor binding regions and amphipathic helices indicated.
  • Figure 2 shows the DNA and deduced amino acid sequence for a cDNA insert encoding the N-terminal region of human apolipoprotein B through amino acid 266.
  • Figure 3 shows the DNA sequence and deduced amino acid sequence for the N-terminal region of human apolipoprotein B through amino acid 430.
  • Figure 4a shows the base sequence of DNA used in the linking region of a fusion protein containing portions of apoB and apoAI;
  • Figure 4b diagrams the portions of apoAI genoraic DNA used.
  • Figure 5 shows the. DNA sequence and deduced amino acid sequence of a fusion protein containing apoB receptor binding and apoAI amphipathic regions.
  • LDL receptor binding region refers to a single peptide sequence which is capable of binding to LDL receptors.
  • a protein fits the definition of containing an LDL receptor binding region if it is capable of competing with iodine-125 labelled LDL in binding to surface receptors on human fibroblasts as described by Goldstein, J.L., et al. Cell (1976) 1_: 85-95 or to receptors on isolated liver membranes as described by Basu, S.K., et al, PNAS (1976) 73: 3178-3182.
  • binding regions In general, they appear to contain sectors of positive charge, generally characterized as segments of the protein containing multiple lysine (or arginine) residues in relatively exposed portions of the protein. Segments containing these residues are often bracketed by cysteine residues, thus providing substrates for disulfide linkages to consolidate the exposure of the positive charges at the surface.
  • concentrations of lysine and arginine residues occur at positions 18-24, 87-90, 166-169, and 225-228. Cysteine residues at positions 12, 51. 61. 70. 159, 185. 218.
  • the receptor binding region of apoE has been determined by others to reside in the region of amino acid residues 140-160 (McClean, J.W., et al, supra). This region contains a high concentration of arginine residues as well as two lysines.
  • a "positively charged domain" as used in describing a sector of an LDL receptor binding region refers to a sequence of four consecutive amino acids bearing, at neutral pH. at least three positive charges. Amino acid side chains which confer positive charges at neutral pH are arginine and lysine.
  • a "lipophilic" protein as used herein refers to a protein capable of binding phospholipids or other lipids such as cholesterol, cholesteryl esters, or triglycerides to form protein lipid complexes, as measured, for example, by their circular dichroic (DC) spectra (see Sparrow, J.T.. et al, supra and Morrisett, et al, Ann Rev Biochem (1975) 4: 183-207). The capacity of proteins to complex with lipids has been characterized fairly extensively.
  • the amphipathic ⁇ helix contains a sequence of amino acids such that an ⁇ helix conformation is stabilized and such that the hydrophobic side chain amino acids reside on one face of the helix while the hydrophilic and charged side chain amino acids reside on the opposite face.
  • the helix presents a hydrophilic face and hydrophobic face. This permits association of lipids with the hydrophobic face while solubilizing the entire complex through the hydrophilicity of the other.
  • operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences operably linked to coding sequences are capable of effecting the expression of the coding sequence.
  • Control sequence refers to a DNA sequence or sequences which are capable, when properly ligated to a desired coding sequence, of effecting its expression in hosts compatible with such sequences.
  • control sequences include promoters in both procaryotic and eucaryotic hosts, and in procaryotic organisms also include ribosome binding site sequences, and, in eucaryotes, termination signals. Additional factors necessary or helpful in effecting expression may subsequently be identified.
  • control sequences simply refers to whatever DNA sequence may be required to effect expression in the particular host used.
  • Cells or “recombinant host cells” or “host cells” are often used interchangably as will be clear from the context. These terms include the immediate subject cell, and, of course, the progeny thereof. It is understood that not all progeny are exactly identical to the parental cell, due to chance mutations or differences in environment. However, such altered progeny are included when the above terms are used.
  • Protein and “protein” are used interchangably herein, without regard to chain length.
  • “Derived from” does not necessarily refer to physical derivation. It means, in the case of peptides, that the amino acid sequences correspond to those of a referenced protein; in the case of a DNA that the base sequence corresponds to that of a referenced oligonucleotide, and so forth.
  • Active substance or “ingredient” refers to a material which exerts an intended effect on a subject organism. It may, for example, be therapeutic, regulatory, toxic, or nutritive.
  • compositions of the invention contain
  • a lipophilic protein containing at least one amphipathic ⁇ helical region covalently bound to a target-specific peptide or (c) both a peptide sequence which is an LDL receptor binding region and a lipophilic protein containing at least one amphipathic ⁇ helical region covalently bound to a target specific peptide associated with an active ingredient.
  • B.1 LDL Receptor Binding Region For those embodiments which contain the peptide sequence which is an LDL. receptor binding region, this sequence is covalently bound to a substance which is either itself an active ingredient in a pharmaceutical or nutrient composition or is a carrier for such active ingredients.
  • One embodiment of the active substance covalently bound to the LDL receptor binding region is an additional polypeptide fused to the binding region which is produced by construction, at the DNA level, of a nucleotide sequence encoding the entire fusion protein. While conjugation at the protein level is theoretically possible, it is difficult to accomplish using standard techniques since the LDL binding region itself contains the side chain amino groups and the cysteine residues ordinarily used as functional groups in binding to linkers conventionally used in formation of, for example, immunoconjugates. To the extent that peptide linkers specific for carboxy terminal groups on the LDL receptor region can be found, covalent binding at the protein level is feasible.
  • Any polypeptide whose amino acid sequence is known, or for which a cDNA or genomic clone exists can be used as the active ingredient.
  • Such peptides include, for example, human growth hormone, urokinase, tumor necrosis factor, ⁇ -, J3-, and ⁇ -interferons, various toxins such as ricin toxin, ricin A chain, diphtheria toxin, PAP, abrin, gelonin, and the like, insulin, various growth factors, hormones such as LHRH, and so forth.
  • Any biologically active peptide for which it is possible to construct a DNA sequence encoding primary structure is suitable for fusion to the DNA -TL3-
  • Additional active ingredients which are not peptides, but which are reactive or can be made reactive with the carboxyl group of the LDL receptor binding peptide may also be directly covalently linked to the binding region peptide.
  • the carrier may be the lipophilic protein bearing an amphipathic ⁇ helical domain as is described in the illustration below.
  • Additional protein carriers include human serum albumin which is especially suitable for water soluble compounds.
  • the role of the albumins in drug transport is already well established, and the DNA sequence encoding human serum albumin (HSA) is known. Therefore, HSA encoding DNA may be fused to the LDL receptor binding region encoding DNA to produce a sequence encoding a fusion in which the HSA acts as the carrier moiety.
  • the active ingredient in this case is generally hydrophilic. and includes such conventional drugs as Warfarin, penicillinase resistant penicillins, and sulfona ides.
  • DNA sequence encoding the desired fusion protein with active peptide ingredient or carrier utilizes standard restriction endonuclease mediated cleavage techniques, techniques for modification of termini, as required, such as blunt ending, removal of nucleotide sequences, ligation of linkers, and the like, and religation of the components of the sequence.
  • the portions of the DNA sequence are obtained either using synthetic techniques such as those employing commercially available DNA synthesizers*in combination with annealing of overlapping single strands, filling in with DNA polymerase, and ligation of portions of the sequences, or parts may be derived from cDNA or genomic clones.
  • control sequences compatible with procaryotes and eucaryotic hosts are also understood.
  • a number of control sequences usable in expressing the coding sequence for the fusion proteins of the invention are set forth in the section on Standard Methods contained hereinbelow. This section also summarizes standard procedures for transformation and expression of these systems in a variety of exemplary hosts.
  • amphipathic domain can be conjugated at the DNA or protein level as appropriate to other specific receptor binding moieties such as, most preferably, imraunoglobulins as well as to the LDL-receptor binding region.
  • Techniques for conjugation of proteins into conjugate immunotoxins at the protein level are well understood in the art.
  • Commercially available linkers include the popular SPDP which results in an amide linkage to a side chain amino group at one terminus of the linker and a sulfhydryl at the other, as well as linkers forming thioethers at this terminus.
  • linkers having different functionalities at either terminus are now well known in the art.
  • the active esters for example succinimidyl esters, of 6-maleimidocaproic or 2-iodoacetic acid.
  • the amphipathic helical region of the lipophilic protein is used as a carrier for the active ingredient in the composition.
  • the active ingredient may simply itself be a lipid, such as, for example, Intralipid, a fat soluble vitamin composition microeraulsion.
  • the microemulsion may also contain additional active ingredients, such as fat soluble drugs including vitamins and chemotherapeutic agents as is described in U.S. serial no. 765,359, filed 13 August 1985, assigned to the same assignee and incorporated herein by reference.
  • the lipophilic protein serves not as an active ingredient itself, but rather as a carrier to support this active ingredient.
  • the binding region may be fused either at the protein or the DNA level.
  • the LDL receptor binding moiety is used in this regard and fusion at the DNA level is preferred.
  • conjugation at the protein level may also be convenient.
  • the lipophilic portion behaves as carrier in a manner described as above for a suitable active hydrophobic ingredient.
  • compositions of the invention are administered as pharmaceutical compositions in a conventional manner. They may be administered parenterally, for example, intravenously or subcutaneously. With proper formulation, it may be possible to administer them as oral dosages.
  • the dosage level is, of course, highly dependent on the nature of the active ingredient, and must be calculated based on the efficacy of this ingredient, as well as on the usual parameters relating to the nature of the subject and condition to be treated.
  • the delivery compositions of the invention are formulated as unit dosages in injectable form,- such as an emulsion, suspension or solution, in association with at least one standard excipient, such as water, dextrose solution, saline solution, and the like.
  • excipient such as water, dextrose solution, saline solution, and the like.
  • the LDL receptor binding region is derived from amino acids 1-250 of apoB protein and the two lipophilic amphipathic helical domains extending through amino acid 402 of this sequence are retained.
  • This apoB derived peptide is itself exemplary of the composition of the invention.
  • this portion is fused through a tripeptide linker to the lipophilic domains of apolipoprotein Al representing the C-terminal 223 amino acids of apoAI.
  • a 17 amino acid peptide representing the sequence from Thr to Ser of apoB as shown in Figure 1 was prepared using standard solid phase peptide synthesis with a commercial synthesizer. The product. judged to be more than 98% pure by HPLC analysis, was used to prepare antisera.
  • High titer antisera against the 17 amino acid peptide was assayed for its ability to inhibit the binding of LDL to its receptor on intact fibroblasts according to the method of Goldstein, J.L., et al. Cell (1976) 1_: 85-95 and to the receptors on bovine liver membranes (Basu, S.K., et al. PNAS (1976) 73.: 3178-3182). The antisera were successful in inhibiting this binding.
  • a 192-fold degenerate 23 base oligonucleotide probe which encodes, taking account of codon redundancy, the first 8 amino acids of the previously determined sequence of apoB-26 was used as a probe.
  • the probe was 5' end labelled with T polynucleotide kinase (PL Biochemicals) and ⁇ 32-PATG, added to the filters and incubated for 14 hr at 50°C.
  • the filters were washed twice at room temperature in 5 x NaCl/Cit, 0.1% SDS, 0.05% sodium pyrophosphate for 15 min and once at 50°C for 20 min. dried and autoradiographed with intensifying screens.
  • LB25-1 One positive plaque, designated LB25-1, was purified and the cDNA insert was subcloned in both orientations into M13/mp8 for sequencing.
  • the nucleotide sequence of this 970 bp insert is shown in Figure 2, along with the deduced amino acid sequence of that reading frame which agrees with the amino terminal sequence determined from the B-26 peptide.
  • the sequence of LB25-1 contains an open reading frame extending 800 nucleotides downstream encoding 30 kd of protein, and analysis of the predicted protein sequence directly upstream suggests the presence of a hydrophobic signal sequence preceded by a methionine residue.
  • the EcoRI insert was subcloned into ⁇ BR322 to obtain pB25-l for amplification. pB25-l thus contains some 5' untranslated region, the signal sequence, and the first 266 amino acids of the mature protein.
  • LB2A-2 linearized denatured pB25-l insert as probe.
  • LB2A-2 linearized denatured pB25-l insert was purified and partially sequenced as described above. The approximately 2 kb insert was then subcloned into pBR322 to obtain pB25-2 to provide additional downstream apoB sequence.
  • Binding Region/Lipophilic Region DNA encoding the LDL binding region, which is contained in the 266 amino acid encoding sequence of the pB25-l insert and a lipophilic region containing two domains of amphipathic ⁇ helical sequences which is contained in the sequence extending from 267-433 of the mature protein was prepared from pB25-l and pB2A-2.
  • the resulting 2550 bp insert fragment was again isolated by digestion with EcoRI followed by purification using agarose gel electrophoresis.
  • the purified insert was treated with XhoII and the EcoRI/XhoII fragment containing the 5' region of the apoB cDNA subcloned into EcoRI/Ba HI digested pUC8.
  • the EcoRI/Sall apoB encoding insert of 1fD.3 was ligated to a synthetic oligonucleotide designed as a Sall/Smal linker region.
  • the nucleotide sequence of the linker and the relevant amino acid sequence encoded are shown in Figure 4a.
  • the linker was constructed from oligomers 1-6 shown in the figure using standard annealing and ligation techniques.
  • the linker DNA provides codons for an Arg-Arg-Gln tripeptide linker and tracks the DNA sequence extending through the end of the apoAI third exon, into the intron to the Smal site, i.e., the last 3 2/3 codons in exon 3 of the apoAI gene, the exon/intron boundary, and 44 nucleotides of intron 3.
  • the ligated fragment was digested with EcoRI and Smal. and subcloned into EcoRI/S al digested pUC8.
  • the resulting plasmid was partially digested with PstI (which cuts just downstream of Smal) and completely with Smal and ligated to the Stul/Hgal (apoAI fragment 1) and Hgal/PstI (apoAI fragment 2) fragments, described below, to obtain a plasmid containing the DNA sequence shown in Figure 5.
  • Figure 5 shows the DNA sequence encoding the apoB/AI fusion, along with the deduced amino acid sequence.
  • This plasmid, designated pB/AI was used as a source for the DNA sequence encoding the fusion protein containing LDL receptor binding region and some lipophilic region from apoB, and additional lipophilic region from Al.
  • apoAI gene fragments were prepared as follows and are shown in Figure 4b.
  • pPSAI was isolated from the ⁇ genomic clone LAI.12 as disclosed by Seilhamer, et al, (1984) supra, and contains most of the apoAI gene.
  • the plasmid was cut with PstI and the 2.2 kb fragment isolated.
  • the purified insert was then cut with Hgal and the Hgal/PstI fragment (fragment 2) isolated.
  • Another sample of the purified insert was also cut with StuI and Hgal and Stul/Hgal fragment (fragment 1) isolated.
  • B5' and B/AI Fusions The DNA segment encoding the apoB LDL-receptor binding and lipophilic regions (B5 1 ) is used to produce the recombinant protein in a variety of hosts. However, expression in mammalian systems is favored as the host is capable of post translational processing analogous to that experienced by the natively produced protein. Either uninterrupted or genomic sequences including the foregoing DNA encoding the B/AI fusion may be used, as the host is also capable of processing introns.
  • DNAs encoding the compositions of the invention as prepared in 1-TD.3 or D.4 are excised with EcoRI and Sail, or with EcoRI and PstI respectively, provided with EcoRI linkers at the Sail or PstI sites and ligated into pHSl, a host expression vector described- below.
  • the resulting expression vectors are transformed into Chinese Hamster Ovary cells and the cells induced as described below. High producing clones are identified by screening with appropriate antibodies. For the B5' fragment of 1fD.3, antibodies raised against this fragment or the LDL-receptor binding regions thereof are used. For the apoB/AI fusion clones showing positive screening against both anti-apoB and anti-apoAI are selected.
  • pHSl the Host Expression Vector
  • the plasmid pHSl contains 840 bp of the hMT-II sequence from p84H (Karin. M.. et al. Nature (1982) 299: 297-802) which spans from the Hindlll site at position -765 of the hMT-II gene to the BamHI cleavage site at base + 70.
  • Plasmid p84H was digested to completion with BamHI, treated with exonuclease Bal-31 to remove terminal nucleotides, and then digested with Hindlll.
  • the desired 840 bp fragment was ligated into pUC8 (Vieira, J., et al.
  • pHSl contains the hMT-II control sequences upstream of a polylinker containing convenient restriction sites including an EcoRI site to place an inserted DNA into operable linkage with the promoter.
  • E. coli MC1061 to Amp Successful transformants are screened by restriction analysis, and a strain containing the desired plasmid, pMT-B5' or pMT-B/AI, is further propagated to prepare quantities of plasmid DNA.
  • CHO-Kl cells are grown on medium composed of a 1:1 mixture of F12 medium and DME medium with 12% fetal calf serum.
  • the competent cells are co-transformed with pMT-B5' or pMT-B/AI and pSV2:NEO (Southern, P., et al, J Mol Appl Genet (1982) l.: 327-341).
  • pSV2:NEO contains a functional gene conferring resistance to the neomycin analog G418.
  • the cells which have been shown to produce the B5' or B/AI fusion under su-itable conditions, are seeded at 1/10 confluency in basal medium supplemented with 10% fetal calf serum, incubated overnight, and then induced for protein production by addition of zinc chloride in
  • the B5' or B/AI fusion secreted into the medium can be further purified according to the procedures set forth for the amphipathic helix- containing protein summarized below by standard methods known in the art.
  • Proteins containing an amphipathic helix can be readily complexed with lipids.
  • the resulting lipoprotein complex can be separated readily from nonlipid binding proteins on the basis of either size or density as follows.
  • the lipoprotein complex is a particulate complex composed of lipid particles — either emulsion, liposomal, or lipid-disc particles — with associated apolipoprotein.
  • the lipoprotein particles are in the general size range of between about 0.01 to 0.55 ⁇ , and as such, can be readily separated from smaller, soluble proteins by particle exclusion in molecular sieve chromatography.
  • the molecular-sieve material is preferably selected to exclude material in the range 500.000 daltons or greater, insuring that essentially all of noncomplexed proteins will be included (retarded) by passage through the chromatography column bed.
  • the larger lipoprotein complex, which is excluded, will elute in the void volume of the column.
  • the eluted fractions can be monitored, conventionally, by UV spectroscopy or the like.
  • Preferred molecular-sieve material includes Agarose 10%, whose exclusion size is about.1.000,000 daltons.
  • the lipoprotein complex is preferably separated from nonlipid binding peptides by flotation. Typically this is done by adjusting the density of the peptide-mixture medium to between about 1.06-1.21 by addition of a mono- or disaccharide, such as sucrose, or a salt, such as KCl. The medium is then placed in a centrifuge tube, overlayered with a lower density medium, and centrifuged until the complex has concentrated at the top of the tube.
  • the lipoprotein material removed from the gradient may be resuspended in the same density medium and separated a second time, to further remove nonlipid-binding proteins.
  • the lipoprotein complex will separate on its own by flotation if allowed to stand undisturbed in a medium of sufficient density. This flotation procedure is, of course, advantageous in large-scale preparation, since both chromatography and centrifugation steps are eliminated.
  • the apolipoprotein associated with the separated lipoprotein complex may be further purified, as necessary, by delipidating the complex and purifying the peptide by conventional purification methods.
  • INTRALIPID obtained from Cutter Labs (Berkeley, CA) can be used as the substrate to bind the apolipoprotein ligand.
  • INTRALIPID is an artificial lipid emulsion composed of soybean triacylglycerol and egg lecithins. The mesophase, or phospholipid-rich portion of the emulsion is removed by ultracentrifugal flotation in a discontinuous sucrose gradient to obtain the triglyceride-rich emulsion which is at the top of the gradient. The apolipoprotein is incubated with purified INTRALIPID and centrifuged as described for INTRALIPID to obtain the top fraction. The apolipoprotein is recovered by delipidation if desired.
  • procaryotic and eucaryotic systems may be used to express the DNA sequences encoding the recombinant proteins useful in the invention; procaryotic hosts are, of course, the most convenient for cloning procedures. Procaryotes most frequently are represented by various strains of E. coli; however. other microbial strains may also be used. Plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used; for example. E. coli is typically transformed using derivatives of pBR322. a plasmid derived from an E. coli species by Bolivar, et al. Gene (1977) 2.:95.
  • pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides additional markers which can be either retained or destroyed in constructing the desired vector.
  • Commonly used procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator-, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al.
  • eucaryotic microbes such as yeast
  • yeast may also be used as hosts.
  • Laboratory strains of Saccharomyces cerevisiae. Baker's yeast are most used although a number of other strains are commonly available.
  • Vectors employing, for example, the 2 ⁇ origin of replication of Broach, J. R., Meth Enz (1983) 101:307. or other yeast compatible origins of replications (see, for example, Stinchcomb, et al. Nature (1979) 282:39, Tschempe, et al. Gene (1980) 10.:157 and Clarke, L. et al. Meth Enz (1983) 101:300) may be used.
  • Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess, et al, J Adv Enzyme Reg (1968) 7_:149; Holland, et al. Biochemistry (1978) 17:4900) . Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase (Hitzeraan, et al, J Biol Chem (1980) 255:2073) , and those for other glycolytic enzymes.
  • promoters which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochro e C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. It is also believed terminator sequences are desirable at the 3' end of the coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. It is also, of course, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms. See, for example. Axel, et al, U.S. patent 4,399,216.
  • Useful host cell lines include VERO and HeLa cells, and Chinese hamster ovary (CHO) cells.
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and late promoters from Simian Virus 40 (SV 40) (Fiers, et al. Nature (1978) 273:113). or other viral promoters such as those derived from polyoma.
  • Adenovirus 2. bovine papiloma virus, or avian sarcoma viruses.
  • the controllable promoter, hMT-II The controllable promoter, hMT-II (Karin, M.
  • transformation is done using standard techniques appropriate to such cells.
  • the calcium treatment employing calcium chloride, as described by Cohen, S.N., Proc Natl Acad Sci (USA) (1972) i9.:2110, or the RbCl method described in Maniatis. T. , et al. Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Press, p. 254 may be used for procaryotes or other cells which contain substantial cell wall barriers.
  • the calcium phosphate precipitation method of Graham and van der Eb, Virology (1978) 5_2:546, optionally as modified by Wigler, M. , et al.
  • Transformations into yeast may be carried out according to the method of Van Solingen, P.. et al, J Bact (1977) 130:946 or of Hsiao, C.L., et al, Proc Natl Acad Sci (USA) (1979) 76:3829.
  • Plasmids containing the desired coding and control sequences employs standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art. and the particulars of which are specified by the manufacturer of these commercially available restriction enzymes. See, e.g.. New England Biolabs, Product Catalog.
  • suitable restriction enzyme or enzymes
  • about 1 ⁇ g of plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution; in the examples herein, typically, an excess of restriction enzyme is used to insure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37°C are workable, although variations can be tolerated.
  • cleaved fragments After each incubation, protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods in Enzymology (1980) 65:499-560. Restriction cleaved fragments may be blunt ended by treating with the large fragment of E.
  • coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide ⁇ triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20 to 25°C in 50 mM Tris pH 7.6. 50 mM NaCl, 6 mM MgCl . 6 mM DTT and 5-10 ⁇ M dNTPs.
  • the Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present.
  • selective repair can be performed by supplying only one of the, or selected, dNTPs within the limitations dictated by the nature of the sticky ends.
  • the mixture is extracted with phenol/chloroform and ethanol precipitated. Treatment under appropriate conditions with SI nuclease or Bal-31 results in hydrolysis of any single-stranded portion.
  • Synthetic oligonucleotides are prepared by the method of Efimov, V.A., et al (Nucleic Acids Res (1982) 6875-6894), and can be prepared using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g.. approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, pH 7.6. 10 mM MgCl . 5 mM dithiothreitol. 1-2 mM ATP, 1.7 pmoles ⁇ 32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
  • Ligations are performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl_, 10 mM DTT, 33 ⁇ g/ml BSA. 10 mM-50 mM NaCl, and either 40 ⁇ M ATP.
  • 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 1 mM ATP.
  • 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation).
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration). Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at 1 ⁇ M total ends concentration.
  • vector fragment In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate and prevent religation of the vector.
  • BAP bacterial alkaline phosphatase
  • CIP calf intestinal alkaline phosphatase
  • Digestions are conducted at pH 8 in approximately 150 mM Tris, in the presence of Na and Mg using about 1 unit of BAP or CIP per ⁇ g of vector at 60° for about one hour.
  • the preparation is extracted with phenol/chloroform and ethanol precipitated.
  • religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.
  • site specific primer directed mutagenesis is used for portions of vectors derived from cDNA or genomic DNA which require sequence modifications. This is conducted using a primer synthetic oligonucleotide complementary to a single stranded phage DNA to be mutagenized except for limited mismatching, representing the desired mutation.
  • the synthetic oligonucleotide is used as a primer to direct synthesis of a strand complementary to the phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. Cultures of the transformed bacteria are plated in top agar, permitting plaque formation from single cells which harbor the phage. Theoretically, 50% of the new plaques will contain the phage having, as a single strand, the mutated form; 50% will have the original sequence. The resulting plaques are hybridized with kinased synthetic primer at a temperature which permits hybridization of an exact match, but at which the mismatches with the original strand are sufficient to prevent hybridization. Plaques which hybridize with the probe are then picked, cultured, and the DNA recovered.
  • Plasmids from the transformants are then prepared according to the method of Clewell, D.B., et al, Proc Natl Acad Sci (USA) (1969) £2.:1159, optionally following chloramphenicol amplification (Clewell, D.B., J. Bacteriol (1972) 110:667) .
  • the isolated DNA is analyzed by restriction and/or sequenced by the dideoxy method of Sanger. F.. et al, Proc Natl Acad Sci (USA) (1977) 74.:5463 as further described by Messing, et al. Nucleic Acids Res (1981) 9.:309, or by the method of Maxam, et al. Methods in Enzymology (1980) 65:499.
  • cDNA or Genomic Library Production Human genomic libraries are constructed in ⁇ phage as is known in the art. See, e.g., Maniatis, T., et al. Cell (1978) 15.: 687-701. cDNA libraries can be prepared as described above in ⁇ gtll phage, or double-stranded cDNA synthesized from mRNA isolated using standard techniques can be prepared for insertion into a plasmid vector such as pBR322 using homopolymeric tailing mediated by calf thymus terminal transferase
  • First strand cDNA is synthesized by the RNA-dependent DNA polymerase from Avian Myeloblastosis Virus, by priming with oligo (dT) 12-18 on 5 ⁇ g mRNA. The RNA template is then liberated from the nascent DNA strand by denaturation at 100°C for 5 min, followed by chilling on ice. Second strand DNA is synthesized by using the large fragment of DNA polymerase I of E. coli. relying on self-priming at the 3 '-end of the first strand molecule, thereby forming a double-stranded hairpin
  • SI nuclease digestion of the double-stranded cDNA takes place in 300 mM NaCl, 30 mM NaOAc. pH 4.5, 3 mM ZnCl for 30 min at 37°C with 600 units enzyme.
  • the cDNA is extracted with phenol:chloroform, and small oligonucleotides are removed by three ethanol precipitations in the presence of ammonium acetate.
  • cDNA roughly greater than the desired length, e.g., 300 base pairs is retained and recovered by precipitation with 70% ethanol.
  • Short (10-30 nucleotides) polymeric tails of deoxycytosine are added to the 3' termini of the cDNA using a reaction containing 0.2 M potassium cacodylate, 25 mM Tris, pH 6.9, 2 mM dithiothreitol. 0.5 mM CoCl-.
  • the reaction is extracted with phenol:chloroform, and small oligonucleotides are removed with three ethanol precipitations in the presence of ammonium acetate.
  • the tailed cDNA is annealed with a host vector such as pBR322 which has been cleaved with, for example, PstI and tailed with oligo dG.
  • a host vector such as pBR322 which has been cleaved with, for example, PstI and tailed with oligo dG.
  • 2.5 ⁇ g pBR322-dG DNA is annealed with the cDNA at a vector concentration of 5 ⁇ g/ml, and the hybrids are transferred into E. coli MC1061 by the
  • cDNA or genomic libraries may be screened if desired using the colony or plaque hybridization procedures. Colonies or plaques are replicated onto duplicate nitrocellulose filter papers (S & S type BA-85) and colonies are allowed to grow at 37°C for 14-16 hr on L agar containing 15 ⁇ g/ml tetracycline.
  • the colonies are lysed with- 10% SDS and the DNA is fixed to the filter by sequential treatment for 5 min with 500 mM NaOH/1.5 M NaCl, then 0.5 M Tris HCKpH 8.0)/1.5 M NaCl followed by 2 x standard saline citrate (SSC). Filters are air dried and baked at 80°C for 2 hr. For nick-translated probe, the duplicate filters are prehybridized at 42°C for 16-18 hr with 10 ml per filter of DNA hybridization buffer (50% formamide (40% formamide if reduced stringency). 5 x SSC.
  • Samples are hybridized with nick-translated DNA probes at 42°C for 12-36 hr for homologous species and 37°C for heterologous species contained in 5 ml of this same DNA hybridization buffer.
  • the filters are washed two times for 30 min, each time at 50°C, in 0.2 x SSC, 0.1% SDS for homologous species hybridization, and at 50°C in 3 x SSC, 0.1% SDS for heterologous species hybridization. Filters are air dried and autoradiographed for 1-3 days at -70°C.
  • the duplicate filters are prehybridized at 42°C for 2-8 hr with 10 ml per filter of oligo-hybridization buffer (6 x SSC, 0.1% SDS, 1 mM EDTA, 5x Denhardt's, 0.05% sodium pyrophosphate and 50 ⁇ g/ml denatured and sheared salmon sperm DNA) .
  • the samples are hybridized with kinased oligonucleotide probes of 15-30 nucleotides under conditions which depend on the composition of the oligonucleotide. Typical conditions employ a temperature of 30-42°C for 24-36 hr with 5 ml/filter of this same oligo-hybridization buffer containing probe.
  • the filters are washed two times for 15 min at 23°C, each time with 6 x SSC, 0.1% SDS and 50 mM sodium phosphate buffer at pH 7, then are washed once for 2 min at the calculated hybridization temperature with 6 x SSC and 0.1% SDS, air dried, and are autoradiographed at -70°C for 2 to 3 days.
  • the DNA for insertion into the host vectors of . the invention may be obtained either by synthetic means, or, if vectors containing such sequences are on deposit or available, by cloning such vectors.
  • alternating sense and anti-sense overlapping single stranded oligonucleotides are prepared, and the alternating sense and anti-sense single stranded portions filled in enzymatically by treating with DNA polymerase and dNTPs.
  • the oligomers are prepared by the method of Efimov, V.A.
  • oligonucleotide synthesizers can be prepared using commercially available automated oligonucleotide synthesizers. Kinasing of single strands prior to annealing or for labeling is achieved using an excess, e.g., approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris. pH 7.6, 10 mM MgCl . 5 mM dithiothreitol, 1-2 raM ATP, 1.7 pmoles ⁇ 32P-ATP (2.9 mCi/mmole), 0.1 mM spermidine, 0.1 mM EDTA.
  • an excess e.g., approximately 10 units of polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris. pH 7.6, 10 mM MgCl . 5 mM dithiothreitol, 1-2 raM ATP
  • Hosts Exemplified Host strains used in cloning and expression herein are as follows:
  • E. coli strain MC1061 was used for cloning and sequencing, and for expression of construction under control of most bacterial promoters.
  • the cells used for mammalian expression are

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Abstract

Les compositions pharmaceutiques ci-décrites dans lesquelles le système d'apport médicamenteux permet d'atteindre sélectivement la cible tirent profit des spirales amphipathiques des apolipoprotéines ou des régions de liaison des apolipoprotéines pour des récepteurs LDL ou les deux. Dans un mode de réalisation, la région de liaison d'un récepteur LDL d'une apolipoprotéine, en particulier apoB ou apoE est lié de manière covalente soit à un ingrédient actif per se, soit à un support pour l'ingrédient actif. Ce système d'administration ou d'apport cible de manière spécifique des tissus qui portent le récepteur LDL. Dans un autre mode de réalisation, les alpha spirales amphipathiques souvant normalement associées à des apolipoprotéines sont conjuguées aux protéines qui ont comme objectif des tissus spécifiques tels que des immunoglobulines ou, de préférence, les régions de protéines de liaison du récepteur LDL. Ce système fusionné est ensuite associé à un ingrédient actif ou à un support intermédiaire pour celui-ci. Les systèmes d'apport médicamenteux ci-décrits constituent de nouveaux systèmes de ciblage pour apporter des ingrédients actifs à des tissus spécifiques.
EP86906200A 1985-10-03 1986-10-02 Nouveaux systemes d'administration de medicaments a base de lipoproteines Withdrawn EP0238645A1 (fr)

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EP0347915A3 (fr) * 1988-06-23 1991-03-20 The Rockefeller University Méthode et agents inhibiteurs de la synthèse des lipoprotéines de basse densité
JPH05508634A (ja) * 1990-07-05 1993-12-02 アクゾ・エヌ・ヴエー レセプター特異的トキシン結合体
AU658578B2 (en) * 1991-06-26 1995-04-27 Bio-Technology General Corporation Purification of recombinant apolipoprotein E from bacteria
EP0611306B1 (fr) * 1991-11-08 1998-07-08 Somatogen, Inc. Hemoglobines utilisees comme agents administrateurs de medicaments
PT636028E (pt) * 1992-04-03 2004-07-30 Univ California Sistema de fornecimento de polinucleotidos auto-organizado
EP0586094A1 (fr) * 1992-08-03 1994-03-09 Wisconsin Alumni Research Foundation Récepteur soluble de LDL et gène
US5795587A (en) 1995-01-23 1998-08-18 University Of Pittsburgh Stable lipid-comprising drug delivery complexes and methods for their production
US6051429A (en) 1995-06-07 2000-04-18 Life Technologies, Inc. Peptide-enhanced cationic lipid transfections
US6344436B1 (en) 1996-01-08 2002-02-05 Baylor College Of Medicine Lipophilic peptides for macromolecule delivery
GB9620153D0 (en) * 1996-09-27 1996-11-13 Univ Strathclyde Non-naturally occurring lipoprotein particle
WO1998024808A2 (fr) * 1996-12-06 1998-06-11 THE UNITED STATES OF AMERICA represented by THE SECRETARY DEPARTMENT OF HEALTH & HUMAN SERVICES, THE NATIONAL INSTITUTES OF HEALTH Inhibition d'allergies provoquees par l'immunoglobuline e grace a un oligopeptide derive de l'immunoglobuline e humaine
US6635623B1 (en) * 1997-06-13 2003-10-21 Baylor College Of Medicine Lipoproteins as nucleic acid vectors
DE19745950A1 (de) * 1997-10-17 1999-04-22 Dds Drug Delivery Service Ges Arzneistoffträgerpartikel für die gewebespezifische Arzneistoffapplikation
EP1090132A1 (fr) * 1998-06-26 2001-04-11 PPL Therapeutics (Scotland) Limited Procedes d'obtention de polypeptides de recombinaison
EP2298728A1 (fr) 1998-11-12 2011-03-23 Life Technologies Corporation Réactifs de transfection
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KR101111477B1 (ko) 2002-12-03 2012-02-23 블랜체트 록펠러 뉴로사이언시즈 인스티튜트 치료제와 연결된 콜레스테롤을 포함하는 접합체
US7803400B2 (en) 2002-12-03 2010-09-28 Blanchette Rockefeller Neurosciences Institute Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier
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US8252338B2 (en) * 2005-11-10 2012-08-28 The Regents Of The University Of California Synthetic LDL as targeted drug delivery vehicle
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US10525152B2 (en) * 2009-10-09 2020-01-07 Signablok, Inc. Methods and compositions for targeted imaging
MX2012006691A (es) * 2009-12-11 2013-02-26 Res Ct Borstel Nuevos conjugados y composiciones para inmunoterapia y tratamiento antitumoral.
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